Electrophoresis in gels is a well-known procedure for determining the molecular weight of substances such as proteins, amino acids, nucleic acids, peptides and other macromolecules by applying an external electrical potential to a gel containing unresolved macromolecules in an electrophoresis cell and measuring the relative movements of the macromolecules. The molecular weight of a molecular specie can be calculated from a set of standards after obtaining a "Ferguson" plot to establish that the charge density of the unknown substance does not deviate from that exhibited by the standards.
The use of polyacrylamide gel electrophoresis (PAGE) allowed a separation effect based on a sieving effect imparted by control of the gel pore size in a "separating gel" layer, in addition to the separation obtained by differences in electrophoretic mobility. However, molecular weight determination by PAGE was complicated by the wide range of electrophoretic charges possessed by the macromolecules present in the system. It was then discovered that these charge differences could be negated by the addition of sodium dodecyl sulfate (SDS) to the system. Large numbers of SDS molecules associate with each protein or macromolecule; the charge of the SDS molecules imparted to the SDS-macromolecule complex is so large that differences in charge, due to the composition of the macromolecule, are not detectable.
A particularly useful electrophoresis procedure is discontinuous SDS-PAGE developed in 1964 (U.S. Pat. No. 3,384,564). Discontinuous SDS-PAGE involves the use of a multi-phasic (discontinuous buffer system, varying in chemical composition, i.e., change in pH and buffer discontinuities.
Often in discontinuous SDS-PAGE, two separately polymerized layers of polyacrylamide, designated as a "stacking gel" and a "separating gel", are prepared. The stacking gel contains a low polymer concentration and has a relatively large pore size. The large pore size allows the sample to concentrate into tightly packed zones. The separating gel contains higher polymer concentrations and has a relatively small pore size. The polymer is the result of reaction between monomer and co-monomer or cross-linking agent (percent C). The effective pore size of the polymer is an inverse function of "total monomer concentration", percent T, defined as the sum of the concentrations of acrylamide and cross-linking agent. The small pore size provides a restrictive effect and produces resolution of the sample.
The uniqueness of this kind of discontinuous SDS-PAGE consists of its ability to concentrate the sample into a narrow starting zone necessary for good resolution. This is achieved in the stacking gel which differs in ionic composition and pH from the buffers in the electrode vessels.
Concentration of the sample into a narrow starting zone produces good resolution and occurs as an interface develops when the leading ion from the buffer of the stacking gel migrates out while the trailing ion of the electrolyte buffer replaces it, both moving in the same direction. The leading ion is chosen to have a higher effective mobility than the ionic species of the sample, and therefore migrates in front of all other ions. Behind the leading ion other zones form and concentrate. The concentrated macromolecule in the samples appear to the eye as one thin "stack". The concentrated sample "stack" continues to migrate through the stacking gel with no change in characteristics until it encounters a discontinuity in entering the separating gel, either in the nature of the supporting medium, i.e., pore size, or in the buffer, e.g., pH. This change produces the separation of the difference macromolecular species into discrete bands. The overall procedure in discontinuous SDS-PAGE thus involves three stages: (a) stacking; (b) unstacking; and (c) resolution.
The R.sub.f is defined as the distance that each band component has traveled from the top of the separating gel to the center of the band, divided by the distance that the leading front has traveled. "Ferguson" plots of the log of R.sub.f versus the percent T of the separating gel are obtained to check for systematic errors. The molecular weight of a particular macromolecule can be calculated by plotting a function of R.sub.f versus a function of molecular weight for a series of known standards. After measuring the R.sub.f for the unknown, one can read the molecular weight from such standard curve.
To carry out a discontinuous SDS-PAGE, the gels are placed in a chamber containing buffer solutions, and the sample is placed on top of the stacking gel and under the upper electrolyte buffer. After an electrical potential is applied, the sample is electrophoresed. The separated macromolecular bands are then stained for visualization. A tracking dye can be added to help monitor the electrophoresis time and to help in the measurement of the relative distance (R.sub.f) of the bands.
Resolution in SDS-PAGE depends on the separation between any two bands of interest and their respective bands widths. The relative mobilities of the constituents at a given pH are very important as they delineate the borders of the moving boundary which is either to stack or to unstack the protein of interest. At a particular pH the particular ionic species in the buffer system determines their electrophoretic mobilities and the electrophoretic resolution of the different proteins.
Polyacrylamide gels are prepared by polymerizing polyacrylamide monomers with a cross-linking agent in the presence of a gel buffer. Polyacrylamide gels are relatively unstable, causing the gels to deteriorate after relatively short periods of time. There is a need for a stabilized polyacrylamide gel which can be prepared and stored for extended periods of time. There is need for a buffer system that in conjunction with such gels and in the presence of SDS will separate proteins by their molecular size.